The collecting and processing of clinical samples is an important aspect in the identification of specific causative agents of disease in the diagnostic process. General considerations in collecting biological samples for examination and culture include the minimization of external contamination of the sample. Once the sample has been collected, the handling and transport of that sample to the clinical laboratory is also an important aspect of the diagnostic process. All samples, regardless of their type and content, are preferably kept hydrated. Further, oxidative processes and enzymatic destruction of the pathogens within the sample should be prevented.
A variety of general and specialized transport media have been devised for the collection, delivery and delayed processing of clinical samples. See, for example, Amies, Can. J. Public Health, 58:296-300 (1967); Bailey et al., "Specimen Containers and their Transport," 29-32, in Diagnostic Microbiology, The C. V. Mosby Co., St. Louis, Mo. (1978); U.S. Pat. No. 4,529,702. However, it is known that different organisms can have particular requirements in order to retain viability in a collection or transport medium. Certain of these organisms, many of which are clinically significant due to their pathogenicity or resistance to treatments, can present difficulties in retaining viability using a single collection or transport media. Examples of some of these organisms are discussed below.
Chlamydia are non-motile, Gram-negative, obligate intracellular parasites. These microbes have been identified as the causative agents of psittacosis-ornithosis, lympho-granuloma venereum, and trachoma and inclusion conjunctivitis in humans. Chlamydia trachomatis has also been implicated in a pneumonia-type syndrome in infants. Chlamydia has further been identified as the agent most frequently involved in non-specific or nongonococcal urethritis. In studies conducted in England and the United States, Chlamydia trachomatis was isolated from between 30% and 50% of patients with nonspecific urethritis. Bowie and Holmes (1977) in Infectious Diseases, 2nd Ed. (Hoeprich, Paul D., ed.), Harper & Row, Hagerstown, pp. 486-487.
Chlamydia must be grown within a cell and can be cultivated in tissue culture or in the yolk sac of an embryonated egg. Clinical specimens suspected of having the Chlamydia pathogen must therefore be collected in a manner so as to minimize external bacterial contamination that would hinder cell culture growth. Samples suspected of containing Chlamydia are typically stored at -70.degree. C.
Mycoplasmas, which include the genera Mycoplasma and Ureaplasma, are extremely small, free-living bacteria which lack a cell wall. Mycoplasmas are therefore resistant to conventional antimicrobials which act upon the bacterial cell wall, such as penicillin and penicillin derivatives. Organisms classified in the genus Mycoplasma do not hydrolyze urea. The organisms in the genus Ureaplasma include those that hydrolyze urea. Both genera have been implicated as agents in diseases of humans. For example, M. pneumoniae has been reported to cause approximately 20% of the cases of primary atypical pneumonia and bronchitis. Ureaplasma have been associated with nongonococcal urethritis. Other diseases and illnesses suggested to be linked to mycoplasmal infections include rheumatoid arthritis and neurological disorders.
Growth media specific for mycoplasma often include a pH indicator for the identification of acid production and various antimicrobials to destroy hardy commensals and prevent overgrowth of the sample. A transport medium that has been used satisfactorily for swab clinical specimens of mycoplasmas comprises trypticase soy broth with 0.5% bovine albumin (Bailey, supra).
Viral pathogens are many and diverse. A common feature of most viruses however, is that they are preferably grown and propagated in cell culture. Therefore, the collection and handling of clinical specimens is extremely important. Specimens must be collected so as to, inter alia, minimize the possibility of bacterial contamination of the cell cultures.
It is clear that the use of a collection or transport medium that may be optimal for use with one type of organism may not be optimal for use with all of these organisms. A sample suspected of having present one or more of the above pathogenic organisms can therefore require the collection of more than one sample using previously available media. It is therefore desirable to have a single collection or transport medium which can sustain viability of a plurality of organisms. One example of a general support media for a variety of microbial pathogens within a variety of clinical samples is Stuart's medium. Stuart's medium (Stuart et al., supra) is a well-known buffered transport medium which includes the components glycerophosphate to permit minimal multiplication and sodium thioglycollate as a reducing agent to prevent oxidation within the sample. Stuart's medium contains no nutrients. The absence of nutrients retards the growth of commensal organisms within the sample which can multiply and overgrow the less hardy pathogens. However, the absence of nutrients in Stuart's media can be detrimental to the viability of less hardy pathogens.
The presence or suspected presence of fastidious pathogens e.g., mycoplasmas, Chlamydia, or certain viruses, in a clinical sample can require that special care be taken with that sample. Specialized media are known which support the viability of a variety of fastidious pathogens. However, these known media were not able to be used successfully with a wide variety of diagnostic procedures, e.g., membrane enzyme-linked immunosorbent assay (membrane ELISA) or polymerase chain reaction (PCR). A collection or transport media which is compatible with reagents or can be used as a reagent itself in the diagnostic procedures performed on the sample can provide an advantage in the field of microorganism diagnosis. Membrane ELISA utilizes a porous membrane to allow certain particles of a particular size to pass through the membrane enhancing the ability of reactants to come into contact, e.g., the antigen and antibody in the formation of the antigen/antibody complex.
Polymerase chain reaction (PCR) is becoming a valuable technique for organism identification in the clinical and reference laboratory and can require specialized reagents for its successful application to microbial diagnostics. Saikki, R. K. (1989) in PCR Technology, Stockton Press, United Kingdom pp. 7-16. PCR is a process by which selected pieces of DNA are amplified within a sample through the action of the enzyme, DNA polymerase so that organisms can be selectively and specifically identified even when they are present in extremely small amounts. In a clinical diagnostic procedure, primers to a single identifying fragment or sequence of patbogen DNA can be added directly to a clinical sample. If the suspected patbogen is present in the sample, PCR techniques can be used to amplify the patbogen DNA so that it is then easily detectable and its presence confirmed. PCR thus allows a sample to be tested directly for the presence of pathogens without requiring that the pathogen be isolated from the clinical sample and cultured separately before positive identification.
A single medium which can be used in the collection of a clinical sample, transport of the sample, and in the diagnostic procedure, e.g. ELISA or PCR, is therefore an advantageous tool in the collecting and processing of clinical samples. The advantages include not only convenience for the clinician or diagnostician, but can provide an economic benefit in the reduction of redundant sample collection and performance of a plurality of identification procedures.